PROJECT SUMMARY/ABSTRACT Antibiotic resistance is one of the most serious medical challenges of our time. This crisis puts patients at risk of untreatable bacterial infections and threatens major advances of modern medicine that rely on antibiotics (transplants, chemotherapy, etc). There are at least 2 million antibiotic resistant infections each year in the US, leading to over 23,000 deaths [1]. It is estimated that without significant action, worldwide annual mortality due to these infections will reach 10 million by 2050, surpassing the predicted mortality from cancer [2]. Understanding resistance mechanisms is critical to designing novel therapeutics to combat resistant bacteria. Heteroresistance is an enigmatic form of antibiotic resistance in which a bacterial isolate harbors a resistant subpopulation that can rapidly replicate in the presence of an antibiotic, while a genetically identical yet susceptible subpopulation is killed [3, 4]. Not only do many species of bacteria exhibit this form of phenotypic resistance, but it has been reported against different classes of antibiotics. Unfortunately, our understanding of heteroresistance is extremely limited and its relevance during infection has been unclear. Using two clinical isolates of the Gram-negative nosocomial pathogen Enterobacter cloacae, we recently showed that heteroresistance to the last-line antibiotic colistin can cause treatment failure in an in vivo model [4]. Furthermore, one of the isolates harbored a very low frequency resistant subpopulation (<1 in 10,000 cells) that was undetected by clinical diagnostic tests, leading to its incorrect classification as colistin susceptible [4]. Our unpublished national surveillance data reveal that colistin heteroresistance is present in 10% of carbapenem-resistant Enterobacteriaceae (CRE), including 18% of carbapenem-resistant Enterobacter, although the majority of these isolates are incorrectly classified as colistin susceptible. Such misclassification could lead clinicians to prescribe colistin inappropriately, leading to treatment failures. Taken together, these data highlight a largely unappreciated epidemic in which colistin heteroresistance is prevalent, overwhelmingly undetected, and may cause unexplained antibiotic treatment failure in the clinic. We will use a combination of genetics, biochemistry, single cell microscopy, flow cytometry and cell sorting to make foundational insights into heteroresistance. Specifically, we will elucidate the dynamics of the resistant subpopulation within colistin heteroresistant Enterobacter, as well as the molecular mechanism controlling resistance. The knowledge gained from this work will form a paradigm with which to study heteroresistance in other bacteria and against diverse antibiotics. Further, the impact of this research will likely extend to eukaryotes as heteroresistance has been observed in fungi [5, 6] and human cancers [7]. Overall, this work will significantly broaden our understanding of how traits exhibited by a subpopulation of cells (phenotypic heterogeneity) can control physiology. This will represent a critical step in our fight against antibiotic resistant bacteria and will lay the foundation for the discovery of novel therapeutics that alleviate human suffering.